DNA Regulation

8/3/2019 DNA Regulation

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Regulation of Gene

Expression


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Type of gene expression

a. Constitutive expression

Some genes are essential and necessary for life, and thereforeare continuously expressed, such as those enzymes involved inTAC. These genes are called housekeeping genes.

b. Induction and repressionThe expression levels of some genes fluctuate in response tothe external signals.Some genes demonstrate higher expression level once beingactivated. It is called induced expression.On the other hand, some genes are repressed and theirexpression levels are lower. It is called repressed expression.


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1.2 Regulatory Elements

Gene expression is a multiple-level process. Transcription initiation is a key point ofcontrolling gene expression. Basic elements that regulate the transcription

include:a. Special DNA sequencesb. Regulatory proteinsc. DNA-protein interaction and protein-proteininteractiond. RNA polymerase


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For prokaryotic systems:

Operon is composed of structural genes, promoter,

operator, and otherregulatory sequences.

a. Special DNA sequence

Other requlatorysequence

Operator

Promoter Sturctural genes


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The DNA sequence that RNA-pol can bind to and

initiate the transcription.

Promoter

Promoter determines:

1. Which strand willserve as a template.

2. Transcription startingpoint.

3. Strength ofpolymerase binding.

4. Frequency ofpolymerase binding.


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promoter operator structural gene

repressorRNA pol

The DNA sequence adjacent to the structural genesthat the repressor protein can bind to and preventthe transcription of structural genes.

Operator


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structural gene

start

AB

Cis-acting elements isthe special DNA sequence thatcan affect the expression of its own gene.

cis-acting generally means "acting from the

same molecule" (i.e., intramolecular). It may beconsidered the opposite of trans-acting which generallymeans "acting from a different molecule"

For eukaryotic systems:
http://bword//!!ARV6FUJ2JP,molecule/http://bword//!!ARV6FUJ2JP,intramolecular/http://bword//!!ARV6FUJ2JP,trans-acting/http://bword//!!ARV6FUJ2JP,trans-acting/http://bword//!!ARV6FUJ2JP,trans-acting/http://bword//!!ARV6FUJ2JP,trans-acting/http://bword//!!ARV6FUJ2JP,intramolecular/http://bword//!!ARV6FUJ2JP,molecule/


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b. Regulatory proteinsFor prokaryotic systems:

Specific factor: It facilitates the binding of RNA-pol toparticular DNA sequence.

Repressor: It binds to the operator and prevent the transcription,known as negative regulation.

Activator: It associates with DNA near the initiation point,resulting in the increase of RNA-pol binding affinity and theenhancement of the transcription efficiency.

For eukaryotic systems: The regulatory proteins are called transcription factors (TF).

After expression, TF will interact with the cis-acting elements toactivate another genes. Therefore, they are referred to as trans-

acting factors.


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Trans-acting factors

a

A

DNA

mRNA

protein A

bA


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The regulation is implemented through numerous interactions betweencis-acting elements and trans-acting factors.

They are non-covalent bond.

Protein-protein interactions

Proteins may have to interact with each other prior to the DNA binding.

Proteins can form a homo or hetero-dimer form to function properly.

Present in prokaryotes as well as eukaryotes.

c. DNA-protein interactions


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Section 2

Gene Regulation ofProkaryotic Systems


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Common features

Prokaryotic genes are polycistron systems, that is,several relevant genes are organized together toform a transcription unit --- operon.

The majority of gene regulation is negative.Inducers are used to remove the repression.


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Operon is a coordinate unit for the regulation.

Transcription initiation is the key point forregulation. Translation can also be regulated.

2.1 Regulation of Transcription


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AYZOPI

structural gene

permease

-galactosidase

regulatory site

operator

promoterCAP-binding site

regulatory gene

transacetylase

Structure of lacoperon


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Metabolism of

lactose


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Bacteria do not express these three enzymes when glucose isavailable. However, bacteria produce those enzymes if lactose ispresent and glucose is absent.

Inducible expression


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Sequence of lacoperon

lacoperon (TTTACA/TATGTT) is a weakpromoter, and has a basal expression level.

CAP (Catabolite gene activator protein)binding site is at -60 region.

CAP is a homodimer with binding ability toDNA and cAMP.


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Glucose inhibits the formation of cAMP.

When glucose is present, [cAMP] is lower. Only after glucose isexhausted, [cAMP] becomes higher. The CAP-cAMP complex is formed,and this complex binds to the CAP binding site on lacoperon.


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AYZOPI

mRNA

RNA pol

When lactose is absent, no lacgene is expressed.

Situation 1


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lacIgene has its own promoter, and its expression can produce LacIrepressor.

The tetrameric Lacrepressor binds to the lacoperator site Olac.

The binding blocks the RNA-pol moving on DNA template, and no lacZ,lacY, and lacA are expressed.


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AYZOPI

repressor

mRNA

lactoseallolactose

RNA pol

galactosidase

Situation 2

When lactose is present, lacZ,lacY, and lacA genes are expressed.


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The galactosidase is weakly expressed (at the basal level).

When lactose is present, it is converted to allolactose or galactose thatbinds to the repressor.

The repressor can no longer bind to the operator, and lacgene can beexpressed.

Galactose , Allolastose and IPTG are referred to as inducer.


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Inducers


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The lacZYA RNA transcript is very unstable and could be degraded quickly.Therefore, the synthesis of three enzymes will be cease under normalcondition.

Presence of lactose


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AYZOPI RNA pol

When glucose is present, the [cAMP] is low, no CAP-cAMP is formed and

the expression of the lacoperon is still low.

Situation 3


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AYZOPI

CAP

cAMP

RNA pol

When glucose is absent and lactose is present, the CAP-cAMP complex

binds to the CAP site to activate the lacgene.

Situation 4


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O O

OO

RNA pol

lactoseno lactose

RNA pol

RNA pol RNA pol

Coordinate expression

No glucose

Glucose


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2.2 Transcription Attenuation

The trpoperon is one of the constitutive genes expressed at the basallevel.

The structural gene of trpoperon encodes 5 enzymes used for thesynthesis of Trp.

Trpoperon


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The trprepressor gene can be expressed, but it does not bind to theoperator.

When Trp is more than enough, the repressor will form a complex withTrp. The complex binds tothe operator, blocking the synthesis of Trp.


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ABCDEOPR

regulatoryregions

structuralgene

Trp

TrpmRNA

attenuated mRNA

trp L attenuator

Trpoperon


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Attenuation mechanism

In addition to the repressor regulation, trpgene has a fine tuningmechanism called attenuation.

The trpoperon is regulated using attenuation mechanism at the translationlevel.


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Leader sequence


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Possible hairpins

1/2 and 3/4 hairpinstructure

2/3 hairpinstructure


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High Trp concentration


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Low Trpconcentration


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Under the normal conditions, theLexA

geneexpressed to repressor proteins that bind topromoters of other genes and block theirexpressions.

Once the repressors are degraded, the repressed

genes will be expressed.

At the basal level, the normal cell contains about1000 copies ofRecA protein.

2.3 Protein Degradation


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SOS response


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LexA digestion


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When DNA is extensively damaged, DNA replication is halted and the

number of ssDNA gaps increases.

The RecA protein binds to this damaged ssDNA, which activates theproteins coprotease activity.

While bound to ssDNA, the RecA protein facilitates the cleavage ofLexA repressor as well as the inactivation of the LexA repressor.


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P1 H1P2hin H2 repressor

DNA

H2 flagellinHin recombinase

P1 H1P2 hin

H2 repressor

H1 flagellintransposed segment

2.4 Genetic Recombination


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An RNA, with sequence complementary to a specific RNA transcript or

mRNA, whose binding prevents processing of the transcript or

translation of the mRNA.

Antisense RNA


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Antisense RNA


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Section 3

Regulation ofEukaryotic Transcription


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Structural features

Large genome: 3 x 109 bps, 35 000genes

Monocistron

Repeated sequences: differentlengths and different frequencies.

Often inverted repeats Splite genes: separated by introns

and exons alternatively


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Regulation features

1. RNA-pol: 3 forms (I, II, and III) fordifferent RNAs

2. Changes of chromosomal structure

Hypersensitive site

Base modification

Isomer-conversion Histone changes


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3. Positive regulation

4. Transcription and translation areseparated

5. Post-transcriptional modification

6. Regulation through intercellular and

intracellular signals


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3.1 Cis-acting elements

They are specific DNA sequences, each of whichregulates transcription of one or more genes.They usually have consensus sequences.

Promoter: TATA box, CAAT box, and GC box,


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Sequence: TATAAAA Location: - 25 ~ - 30 bp

Function: It is the binding site for TFII D, which is required for RNA

polymerase binding. It controls the veracity and frequency of

transcriptional initiation.

TATA box


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Sequence: GCCAAT

Location: ~ -70 bp

Function: It is the binding site for CTF1 (CAAT-

binding transcription factor) and C/EBP(enhancer binding protein).

CAAT box


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Sequence: GGGCGG

Location: -30 ~ -110 bp

Function: It is the binding site for a protein called Sp1.

GC box


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It is a DNA sequence that can determine thetemporal and spatial specificities of expression andincrease the promoter activity.

enhancer


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It is a negative regulation element.

It will repress the transcription once interactedwith specific proteins.

Silencer


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3.2. Trans-acting factors

They are the proteins that bind indirectly to cis-acting elements and then regulate thetranscription initiation.

The trans-acting factors can be transcriptionfactors (TF).


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transcription factors

General transcription factors

Special transcription factors

Transcription activators

EBP (enhancer binding protein)

Transcription inhibitors


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General structure of TF

DNA-binding domain Activation domain

Protein-protein interaction domain


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Promoter and regulatory proteins


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General structure of TF

CTD of RNA-pol II is an important point ofinteraction with mediators and other proteincomplexes.

Cofactors facilitate the TF assembly.


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Transcription repressor


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3.3 DNA-protein interactions

Regulatory proteins have discrete DNA-bindingdomains of particularstructure, i.e., binding motif.

The AA side chains of regulatory proteins interact with bases of DNAthrough H bonds.


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Yeast activator

protein GCN4

Leucine zipper


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Zinc finger


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Steroid hormone receptor


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Mouse regulatory proteinZif268

Zinc finger


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Helix-loop-helix


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Helix-loop-helix

Human transcription factor MAX


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Helix-turn-helix

Lacrepressor


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Helix-turn-helix

Trprepressor

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DNA Regulation